MOIJXL~LARAND CELLULAR NEIJROSCIENCES 1, lo-19 (1990)

The Thymus Gland as a Major Target for the Central Nervous System and the Neuroendocrine System: Neuroendocrine Modulation of

Thymic P,-Adrenergic Receptor Distribution as Revealed by in Vitro Autoradiography

BIANCA MARCHETTI, MARIA C. MORALE, AND GEORGES PELLETIER*

Department of Pharmacology, Medical School, Uniuersity of Catania, 95125 Catania, Italy; and *MRC Group in Molecular Endocrinology, CHUL, Quebec, Canada GIV 462

Received for publication February l&l990

The existence of a dense structural and hormonal in- nervation of the immune system provides the first link in the interaction among the central nervous system, the endocrine system and the immune system. Our re- cent localization of a specific &adrenergic receptor population in the medulla of the rat thymus gland, un- dergoing important changes in density and distribution during ontogenic development, coupled with a marked sexual dimorphism during maturation, prompted us to study changes in distribution of the thymic &adrener- gic receptor under experimental conditions accompa- nied by marked fluctuations of the sex steroid hormonal milieu, using in vitro autoradiography. Moreover, such changes were correlated with alterations in thymocyte proliferative response and thymus histology. Slide- mounted cryostat sections were incubated with iodocy- anopindolol ([‘251]CYP), a specific ligand for /3-adrener- gic receptors. Labeling was detected by autoradiogra- phy, by exposing the sections to Ultrofilm (LKB). The vast majority of receptors localized in the medullar compartment of the thymus are of the &subtype, with no qualitative changes in the predominant &-receptor population under the different conditions studied. Dur- ing the rat estrous cycle, a significant increase in den- sity of the receptor was observed on proestrous, com- pared to the other phases of the cycle. Castration pro- duced a marked decrease in density and loss of organization of the receptor in the medulla, coupled with a marked hypertrophy of the cortical region and a significant increase in cell-mediated immune response. On the other hand, treatment with estradiol, alone or in combination with progesterone, resulted in a profound stimulation of @-adrenergic receptor density, together with an inhibition of thymus weight, atrophy of the cor- tex, and reduced thymic proliferative capacity. Present results indicate that the thymic /3-adrenergic receptor is dramatically influenced by the sex steroid hormonal milieu and that estrogen is a potent stimulator of recep-

tor density. It is suggested that the steroidal modulation of the &-adrenergic receptor may play a role in the changes of sensitivity of the thymic cells to the chang- ing repertoire of the thymic microenvironment, indi- cating that this receptor population is involved in a neu- roendocrine-immune regulatory loop, thereby provid- ing a functional integration of neural, immune, and endocrine signals. CC 1990 Academic Press, Inc.

INTRODUCTION

With the recognition of intercommunicating path- ways delivering reciprocal information to the three ma- jor integrative bodily systems, the central nervous sys- tem (CNS) and the immune and endocrine systems, a new interdisciplinary research area, termed “neuroim- munoendocrinology,” has recently emerged (see l-3). The common morphological and physiological sub- strates providing the circuitry for crosstalk communica- tion between neuroendocrine and immune cells have lead to the proposal that “a biochemical network, a shar- ing and reciprocity of chemical messengers,” connecting the two physiological systems (l-5) exists. The direct innervation of immune organs by the sympathetic and parasympathetic divisions of the autonomic nervous systems (ANS) provides the first morphological link per- mitting the dialogue between the brain and the immune cells (6-11). In this context, the thymus gland, the cen- tral organ of the immune system constituting the micro- environment for T-lymphocyte differentiation and mat- uration, plays a key role in neuroendocrine-immune intercommunications (12-17). The identification of nor- adrenergic nerve terminals (NA) in very close contact with different cell types, such as macrophages, mast cells, epithelial cells, and thymocytes, and the ultra- structural analyses revealing synaptic-like structures between nerve terminals (6-10) support the hypothesis

1044.7431/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

10

NEIJROENDOCRINE CONTROL OF THE THYMIC &ADRENERGIC RECEPTOR 11

of a neuromodulation exerted upon the secretory activ- ity of the thymic epithelial cells. Then, through the neu- ral noradrenergic innervation of the thymus gland (in the subcapsular area, the deep cortex, and the connec- tive tissue of the organ) (8), a fascinating possibility ex- ists: that a neural message could impinge directly on the cellular thymic elements.

The developing thymocytes have been shown to pos- sess specific &adrenergic receptors and to respond to catecholamines with elevation of the second messenger, adenosine 3’:5’-cyclic monophosphate (CAMP) (18-20), leading to alt,ered expression of T-cell surface alloanti- gens. Moreover, neonatal sympathectomy can result in profound alterations of the adult immune response (see 822). While various studies have described the presence of a o-adrenergic receptor-adenylate cyclase system in lymphocytes, thymocytes, macrophages, and eosino- phils, as well as the ability in vitro of adrenergic agonists and antagonists to alter the cellular response (for review see 7, 8, 22), there are no studies, to our knowledge, de- scribing morphological changes in the adrenergic recep- tor population in the different compartments of the in- tact thymus, in its natural microenvironment, where re- ceptors could be exposed to different concentrations of endogenous amines and colocalized transmitters or thymic hormones and neuropeptides.

The marked changes in [j-adrenergic receptor density and distribution accompanying the in vivo maturation and differentiation of thymocyte function, with a clear sexual dimorphism in receptor organization during sex- ual maturation (23), coupled with the important hor- monally mediated relat,ionships between the t hymus and the reproductive system (see 24)) prompted us t,o investi- gate the hormonal modulation of the /&-adrenergic re- ceptor population in the rat thymus. For this purpose. we have assessed changes in receptor density and distri- bution during the rat estrous cycle and after ovariectomy and steroid treatment, using in llitro autoradiography. Moreover, such changes were correlated with alteration in thymocyte responsil-enrss to proliferative agents and 1 hymus morpholqq.

Sprague~~l>awiey (l‘rl:CD(SD)Br) virgin f’emale rats purchased from Charles River (Calco, Como, Italy) were housed two per cage in a temperature (‘22 _+ %‘C)-con- trolled and light ( 16-h dark cycles: lights on at 0,500 h)- c~ontrolled room and received Purina Rat Chow (Ralston Purina) and water ad libitum. The stage of the estrous cycle was evaluated by daily vaginal smears, and only animals showing at least three consecutive 4-day cycles were used. Special care was taken to avoid any environ- mental change that might influence or stress the ani-

mals. Groups (6 or 7 animals) of estrous, diestrous 1, di- estrous 2, and proestrous rats were killed under basal conditions between 0900 and 1000 h.

Treatments

Virgin female rats were castrated either surgically or chemically, by SC injection, with a potent luteinizing hor- mone-releasing hormone agonist (LHRH-A, D-Trp”- LHRH, 1 pg daily for 3 weeks). Castration (OVX) or sham operation was carried out under light ether anes- thesia. For 3 weeks, OVX animals received twice daily SC injections of the following steroids: 17/3-estradiol (E,, 1.125 pg/rat), progesterone (P, 2 mg/rat), and dihy- drotestosterone (DHT, 250 pg/rat), alone or in combina- tion, in 0.2 ml 1% gelatin-0.9% NaCl. The compounds were first dissolved in a small volume of ethanol before further dilution with 1% gelatin-0.9% NaCl. Groups of intact, sham-operated, and OVX rats received daily in- jections of the vehicle.

Tissue Preparation for Autoradiographic Localization of

@-Adrenergic Receptors

Thymus glands were frozen for 30 set in isopentane chilled over dry ice. Frozen thymus sections were cut to a thickness of 8-10 /*m and mounted onto gelatin-coated microscope slides by thawing. Sections were dehydrated under vacuum at 4°C for 18 h and, if not immediately used, were stored at -70°C. For receptor localization, all slides were processed in parallel, under the same stan- dard conditions (time of exposure, incubation, and de- velopment) (25, 26). The slides were brought to room temperature, preincubated for 30 min in 0.1 M Mg-Tris- HCl buffer, pH 7.6, and incubated with the iodinated tracer in the same buffer in the presence or absence of different adrenergic agonists or antagonists, as de- scribed below. After rinsing, the sections were fixed in 2.5% glutaraldehyde for 10 min, dried at room tempera- ture, and placed against ultrafilm (LKB, Rockville, MD) or coated in liquid chromatographic emulsion (Kodak NTB2, Eastman Kodak, Rochester, NY).

I ne iodinated ligand, ~odoc~a~lopincloloi ii’ ’ IjCYPi, was obtamed tram New England Nuclear (NEN, UK) at a specific activity of 2000 Ci/mmol. Thymic sections were preincubated in 0.1 M Mg-TrissHCl buffer, pH 7.6, and incubat,ed in the same buffer containing 100,000 cpm/ml [“‘IICYP in the presence or absence of lo- ’ M ( )propranolol, as previously described (25, 26). Dis- placement of [“‘I]CYP binding was achieved by the ad- dition of increasing concentrations of I.-propranolol, zinterol (a Y-adrenergic agonist with a preferential affinity for &receptor subtype), or practolol (a /3-adren- ergic antagonist that binds preferentially to fi,-receptor

12 MARCHETTI, MORALE, AND PELLETIER

subtype). Previous time-course studies had indicated that saturation of the receptor occurred after 90 min. In- cubations were then carried out for 2 h to ensure satura- tions of all sites.

IC& values of competition potency for [lz51]CYP were derived from gamma counting of tissue sections scraped off slides. They were calculated using a weighed iterative nonlinear least-squares regression (27). Optical density of autoradiograms (from X-ray films) was measured with a Loats Image Analysis System (Amersham). Twelve thymus sections/thymus (a total of 36 sections/ experimental group) were analyzed. The results which represent optical density units per total thymus section are expressed as means -t SEM.

Preparation of Thymocyte Cultures

Thymic lymphocytes were prepared as previously de- scribed (28). Briefly, after rapid collection under sterile conditions, thymus glands were placed in cold RPM1 1640 containing 1% fetal calf serum (FCS) supple- mented with antibiotics (100 IU penicillin and 100 pg streptomycin). Cells were gently teased apart and passed through a nylon mesh (40-pm mesh) to remove clumps of cells and connective tissue. After two washes with PBS, the cell suspension was adjusted to a final concen- tration of 2 X lo6 cells/ml in RPM1 1640 with 10% FCS in 96-well microtiter plates, and blastogenic assays were carried out with 0.2-ml suspensions. Cell viability was determined by the use of fluorescein diacetate (29). Thy- mocyte cultures were incubated with the mitogen conca- navalin A (Con A, O-2 pg/ml). After incubation for 72 h at 37°C with 5% COP, E3H]thymidine (1 &i/well; 25 Ci/ mmol) was added, and the cultures were incubated for an additional 6 h. Labeled DNA was collected on glass fiber filters using an automatic cell harvester, and radio- activity was determined by liquid scintillation spectro- photometry.

Thymus Histology

Thymus glands were fixed in 10% formalin, and 4-pm sections from paraffin-embedded tissue blocks were ob- tained and stained with hematoxylin and eosin.

Materials

The following compounds were gifts: zinterol (Mead- Johnson) and L- and D-propranolol (Ayerst Research Laboratories, Montreal, Quebec, Canada). Practolol was kindly provided by Dr. M. G. Caron (Durham, NC). Con A was purchased from Pharmacia (Uppsala, Sweden), while the other commercially available adrenergic com- pounds were obtained from Sigma. RPM1 1640, penicil- lin, and streptomycin were obtained from GIBCO (Scot- land, UK) and Omnifluor was from New England Nuclear (Boston, MA).

DIESTRUS 1 DIESTRUS 2 PROESTRUS ESTRUS

FIG. 1. &Adrenergic receptor concentration within the thymus gland of female rats during the estrous cycle. Slide-mounted thymus sections from estrous, diestrous 1, diestrous 2, and proestrous animals were incubated with [iz51]CYP (100,000 cpm/ml) in the presence or absence of an excess (10m6 M) of (-)proparanolol. P-Adrenergic recep- tor levels are expressed as arbitrary optical density units (OD) and represent mean values f SEM. Nonspecific binding reaction had an OD = 0.035 f 0.0003. **P < 0.01 compared to the other phases of the cycle.

Data Analysis

Binding data were analyzed with a Hewlett-Packard calculator (Model 9845, Palo Alto, CA) using a program based on model II of Rodbard and Lewald (27). Statisti- cal significance was assessed according to the multiple range test of Duncan-Kramer (30) and the analysis of variance (ANOVA).

RESULTS

Thymic P-Adrenergic Receptors during the Rat Estrous Cycle

Incubation of [1251]CYP with slide-mounted thymus sections from estrous, diestrous 1, diestrous 2, and pro- estrous animals showed marked differences in receptor density (Fig. 1). As observed, P-adrenergic receptor con- centrations evaluated by densitometry of X-ray films of thymic sections, expressed as arbitrary optical density (OD) units, were significantly (P < 0.01) higher during proestrous (0.78 -I- 0.02) than during estrous (0.50 f 0.02), diestrous 1 (0.52 + 0.02), and diestrous 2 (0.58 k 0.02). In accordance with our previous (23) and other (18) data, our results show that the population of p-ad- renergic receptors of the rat thymus gland is of the pZ- subtype (Table 1).

To determine whether the physiological fluctuations in plasma sex steroids during the estrous cycle could change the predominant &adrenergic receptor popula- tion characterized in the rat thymus during pubertal de- velopment (23), competition studies using subtype selec- tive adrenergic compounds were performed (see 31-33). As observed in Table 1, the present findings confirm the predominant representation of the &adrenergic recep- tor population, with no qualitative variations during the cycle. In fact, at each stage of the ovarian cycle, the most

NEUROENDOCRINE CONTROL OF THE THYMIC &ADRENERGIC RECEPTOR 13

TABLE 1

Specificity of [“51]CYP Binding to the Thymus Gland during the Rat Estrous Cycle

Agonist/antagonist IG,, (144 M)

Agonists Zinterol 2.8 x 10 x t 0.05 (~ )Isoproterenol 4.4 x 10 4 i 0.03 ( )Epinephrine .5.4 x lo-’ If- 0.05 (-)Norepinephrine 6.0 X 1K7 f 0.05

Antagonists ( )Propranolol 5.6 y lo- “’ t- 0.04 Practolol 1 .o x 10~” t 0.05

____.-.-.

Note. Displacement curves were obtained from gamma counts oftis- sue sections scraped off slides, previously incubated with [‘?]CYP ( 100,000 cpm/ml) and increasing concentrations of different adrener- gic agonists or antagonists. The I& values clearly indicate that the vast majority of receptors in the rat thymus are of the &subtype, with no qualitative variations in the predominant &-receptor representa- tion during the estrous cycle.

potent competitor was zinterol, followed by (-)isopro- terenol 2 (-)epinephrine B (-)norepinephrine (31-33). Moreover, the concentration response curves of the un- labeled adrenergic compounds competing for [““I]CYP hinding were parallel (data not shown). The prevalent /‘$ nature of the thymic receptor was further confirmed by the finding that compounds more selective for the flZ- adrenergic receptor (such as zinterol) were more potent in displacing [““I]CYP binding to thymic sections than agents more selective for the B,-adrenergic receptor (such as practolol), at each stage of the ovarian cycle (31 -33) (Table 1).

‘I’h.vmic o-Adrenergic Receptors following Ouariectomy and Treatment with Sex Steroids As observed in Fig. 2, hormonal treatments markedly

influence b-adrenergic receptor concentration, ex- pressed as optical density units. Three weeks after chemical castration (treatment with LHRH-A), recep- tor density from control proestrous levels of 0.68 & 0.05 fall to 0.42 i- 0.04 (P < 0.01). Similarly, OVX produced a significant (P < 0.01) decrease in receptor density to 0.40 -t 0.03. On the other hand, treatment wit.h E, re- yulted in a potent stimulatory effect, increasing /j-adren- rrgic receptor density to 0.70 tr 0.06. While P alone did :?ot modifv receptor concentration (0.43 + 0.04). in com- parison with OVX (0.40 i- 0.03), the combination of E, + P counteracted the castration-induced receptor de- crease. When the androgen, DHT, was administered daily to OVX rats, a slight but not significant increase (0.53 -+ 0.04) in receptor density could be measured (Fig. 2).

Thymic ij-Adrenergic Receptor Localization and Distribution As observed in Fig. 3, the autoradiograms of thymic

sections analyzed under the experimental conditions

studied show marked differences in organization and distribution of the thymic &-adrenergic receptor. When animals are sacrificed on the morning of proestrous, the binding reaction is very high in the thymic medulla, sur- rounded by a much lighter region, the cortex (Fig. 3A). Three weeks after OVX (Fig. 3B), a profound loss of binding and organization of the receptor population was observed, compared to density of proestrous thymus sec- tions or to that measured after sham operation. By con- trast, treatment with estradiol of castrated rats induced a potent stimulation of the binding reaction (Fig. 3C). Figure 3D illustrates a consecutive section where an ex- cess (lo--’ M) of (-)propranolol was incubated in the presence of [““I]CYP (100,000 cpm/ml), showing the nonspecific binding reaction (OD = 0.01 + 0.001).

Figure 4 shows X-ray films from the same experimen- tal groups demonstrating the selective localization of the &-adrenergic receptor in the thymus medulla (darkly stained). It is possible to appreciate the marked differ- ence in receptor density in intact proestrous rats (Fig. 4A) and following hormonal manipulations (Figs. 4B and 4C). The nonspecific binding is also illustrated (Fig. 4D).

Thymus Weight and Morphology

The weight of the thymus gland showed significant al- terations under the different experimental conditions. In accordance with previous investigations (see 24, 34- 36), the weight of the gland increased markedly after chemical or surgical castration (Table 2). By contrast, treatment with E, alone or in combination with P coun- teracted the effect of castration. Daily administration of P alone or DHT did not produce significant effects in the thymus weight of OVX animals. The histological exami- nation of the t,hymus of intact rat,s treated with the LH- RH-A or the castrated animals revealed the well-known

;-I;, j / I i / i-i

OVX OVX*E OVXaP Cb’X*E+P OVX

DHT TREATMENTS

FIG. 2. ,-l,-.4clrenerpic receptors under different hormonal manip ulations. Animals were either treated with an LHRH agonist (I,HRH- A) or castrated and received daily sc injections of the indicated ste- roids. I?; 173.estradiol; P, progesterone; DHT, dihydrotestosterone. Receptor density expressed as optical density units (OD) was calcu- lated from autoradiograms (from X-ray films) and represents the mean I SEM. **P < 0.01 compared to intact proestrous.

14 MARCHETTI, MORALE, AND PELLETIER

FIG. 3. Autoradiographic distribution of the thymic &adrenergic receptor under different hormonal treatments. A Loats Image Analysis System (Amersham) was used to analyze differences in receptor localization. As observed in the right upper corner of each picture, cold colors (including blue, violet, and rose) indicate the absence or low concentration of receptors, while green, yellow, orange, and red colors represent the increasing receptor concentrations. (A) Thymic section from a proestrous animal; note the high concentration of receptor distributed over the medullary regions. (B) Thymic section from a castrated rat (note the dramatic loss of receptor organization in the medulla, together with a profound reduction in receptor density). The hypertrophic effect of castration can also be observed. (C) Effect of treatment of castrated rats with estradiol (note the important receptor stimulation accompanied by thymic atrophy). (D) Nonspecific binding reaction after an excess ( 1O-6) of (-)propranolol. (X2.6)

(see 24, 34, 35) hypertrophy of the cortical region, Thymocyte Proliferative Capacity densely packed with lymphocytes, while treatment with Ez produced a significant atrophy of the gland, charac- Figure 5 shows the effect of the hormonal manipula- terized by a profound reduction of the cortex and atro- tions on the ability of thymocytes to respond to a mito- phy of the lobules, with no significant modification of genie substance, such as Con A (O-2 pg), following cas- the medullar compartment (not shown). tration or treatment with the different sex steroids. As

NEUROENDOCRINE CONTROL OF THE THYMIC &ADRENERGIC RECEPTOR 15

D

FIG. 4. X-ray films showing changes in distribution of’ the thvmic ,l,-adrenergic receptors after hormonal manipulations. (A) Proestrous i~lote the selective localization of the receptor in the thymic medullary compartment (darkly stained)). (H) Castration (note the important fall in recept~lr density). (CI E1f’ec.t ol’estradiol trratment ofcastrwred rats. (1)) Nonspecific hindiny reaction. (r~2.7)

observed, blastogenic activity monitored by [“Hlthymi- dine incorporat,ion following Con A stimulation was markedly increased in thymic cultures from castrat,ed rats. Ry contrast,, treatment of OVX animals with E, sig- nificantly reduced the cell-mediated immune response to levels observed in intact rats. A similar decrease in the proiiferative response was measured following treat ment of OVX rats with all the other steroids tested. Of interest., and in agreement with our previous findings ob- tained in aging animals (%), treatment of intact rats with the LHRH-A produced a profound stimulation of rhe proliferative capacity of thymocytes, with levels of hlastogenic activity significantly (P -C 0.01) higher than those measured in OVX rats (Fig. 5).

DISCUSSION

We have used in uitro autoradiography with the aim of studying the compartment~alization and distribution

of the &adrenergic receptor population of the rat thy- mus gland, following different hormonal manipulations accompanied by marked alterations of thymus morpho- logic appearance and the proliferative capacity of thy- mocytes. The present results clearly indicate that the 13,. adrenergic receptor population of the rat thymus gland, l~~calized predominantly in the medullary compartment. id markedly sensitive to the physiological alterations of the sex steroid hormonal milieu. Such a suggestion per- tains to (a) the significant stimulation of receptor den- sity during the proestrous phase of the cycle, (b) the dra- matic reduction of the binding reaction and the loss of morphological organization of the receptor after castra- tion, and (c) the potent stimulation of receptor density accompanying treatment with estradiol. Moreover, these effects on the &-adrenergic receptor population are paralleled by significant alterations in the prolifera- tive capacity and morphology of the thymus. In view of the sexual dimorphism in both density and distribution

16 MARCHETTI, MORALE, AND PELLETIER

TABLE 2

Thymus Weight under Different Hormonal Manipulations

Experimental group Thymus weight (mg)

Intact proestrous 305 f 32 Intact + LHRH-A 600 +- 49** ovx 628 f 51** OVX + E, 375 k 40 ovx+p 520 f 55** OVX+E,+P 350 -+ 30 OVX + DHT 300 k 28

Note. Animals were treated twice daily with sc doses of the following steroids, 170.e&radio1 (E2, 1.125 pg), progesterone (P, 2 mg), dihy- drotestosterone (DHT, 250 pg), alone or in combination, for 3 weeks. A group of intact rats received twice daily SC injections of the potent LHRH analog D-Trpe-LHRH-ET (LHRH-A, 1 pg).

** P < 0.01 vs intact proestrous.

of the &-adrenergic receptor observed during thymus ontogeny (23), and of the important sex steroid influence on thymocyte function, it seems tempting to speculate that part of the mechanism(s) by which sex steroid hor- mones may feedback information to the thymus gland may involve alterations of the P-adrenergic receptor population.

A functional role for a P-adrenergic receptor coupled with the adenylate cyclase CAMP system in thymocytes is supported by different lines of evidence, including the early sympathetic innervation of the thymus (evident at Day 17 of gestation in mice) (see S), the marked alter- ations in adult immune function (7,8, 22) observed fol- lowing neonatal sympathectomy, the sensitivity of de- veloping thymocytes to catecholamines in proliferative activity, and the expression of surface antigens (E-20). Specific &adrenergic receptors have been characterized in mice thymocytes and isoproterenol was found to stim- ulate CAMP levels, the effect being antagonized by pro- pranolol (18). Our data showing P-adrenergic receptors in the thymic medulla using [ lz51] CYP as radioligand are in agreement with and extend the study of Singh and collaborators (18).

On the other hand, in contrast to the early data of Brodde et al. (37), introducing [1251]CYP as a highly spe- cific P-adrenergic receptor ligand, and the more recent finding of Keshles and Levitzki (38), demonstrating that [ 1251]CYP binds exclusively to /3-adrenergic receptors (whereas 1251-hydroxybenzylpindolo1 binds to both p re- ceptors and serotonin receptors), Schlicker and co- workers (39) have reported that [‘2”I]CYP is a highly po- tent and selective antagonist at the presynaptic seroto- nin autoreceptor in the rat brain cortex. Moreover, Pazos and associates (40) have shown that in some brain areas the binding of [1251]CYP was blocked with high affinity by some serotoninergic ligands (such as RU- 24969 and serotonin), supplying direct evidence for the labeling of a subclass of serotonin receptors (the 5HT-

1B class). While further studies are required to clarify this issue at the thymic gland level, the high potency of the /3,-selective drug (the agonist zinterol) and the low affinity of the P-selective drug, such as practolol, in com- peting for [‘251]CYP binding support the & nature of the thymic gland adrenergic receptor (see 31-33). Such findings are in agreement with the /3,-receptor specificity in rat cerebellum (41), as well as in rat lung (33), ovarian tissue (25, 26), dispersed Leydig cells (42), intermediate lobe of the pituitary gland (43), mammary gland (26), and mammary tumors induced by carcinogen adminis- tration (44).

Results of this study clearly indicate that the thymic /3-adrenergic receptor, a part being a target of the central nervous system, is also a key target of gonadal hormones. The observed changes in both density and distribution of this receptor under the different conditions studied clearly suggest that the known gonadal hormone influ- ence upon the thymus gland might change the sensitiv- ity of the thymic cells to central nervous system input.

The presence of receptors for estrogen and testoster- one on the reticuloepithelial matrix of the thymus pro- vides a substrate for gonadal influence on the thymus (see 36). The thymus, in turn, not only secretes peptides such as thymosin &, which stimulates LHRH secretion from the hypothalamus (47), but is also able to recognize the primary hormone involved in neuroendocrine control of reproductive processes, LHRH (34). Specific low-affinity LHRH binding sites coupled with the prolif- erative activity of thymocytes through an action on the rate-limiting step enzyme of polyamine biosynthesis, or- nithine decarboxylase (ODC), have been recently char- acterized in the rat thymus (28). It seems of interest that

m- lo 60 x E

0.5 1.0 1.5 2.0

Con-A Concentration (pglml)

FIG. 5. Blastogenic transformation of rat thymocytes under different hormonal treatments. Thymocyte proliferative capacity was tested in thymic cell cultures from the indicated groups after stimula- tion with the T-dependent mitogen concanavalin A (Con A, O-2.5 pg/ ml). After incubation for 72 h at 37°C with 5% CO,, [3H]thymidine was added, and cultures were incubated for an additional 6 h. Labeled DNA was collected on glass fiber filters, and radioactivity was deter- mined by liquid scintillation spectrometry.

NEI’ROENDOCRINE CONTROL OF THE THYMIC &ADRENERGIC RECEPTOR 17

the chronic treatment with LHRH-A of intact animals produced a marked effect on the thymic @-adrenergic re- ceptor population accompanied by a significant increase in the weight and proliferative capacity of the thymo- cytes (Fig. 5). Such findings are in agreement with and support our recent, study showing that treatment of aging rats with LHRH-A restores thymus morphology and function (35).

The dramatic involution of the maturing thymus that occurs following puberty in male and female rodents is delayed by prepubertal castration (36,48), while admin- istration of estrogens and androgens can reverse this effect. The sexual regulation of immune function is fur- ther substantiated by the greater production of immuno- globulin in females than in males both in viuo and in vitro. The influence of sex steroids on certain autoim- mune disorders is also well established (36). In this re- spect, an interesting point linking fi-adrenergic recep- t,ors, gonadal hormones, and the immune response is the fact that during its postnatal development the organiza- tion and increase in density of the receptor in the medul- lary thymic compartment occur much earlier and with higher intensity in females than in male animals (23). Moreover, before puberty, incubation of thymocytes from female rats with adrenergic agonists potentiates the blastogenic response to the mitogen concanavalin A (23), an effect that is not observed after puberty. Also at puberty a marked and physiological decrease in the abil- ity of thymocytes to respond to Con A can be observed (23). Our present study shows that in female rats a cyclic variation in density and distribution of the adrenergic receptor accompanies the phase of maximal estrogenic stimulation, while castration alters markedly the organ architecture and receptor distribution, resulting in a profound fall in receptor density. It is then possible that such cyclic changes occurring at each estrous cycle might be involved somehow in the cyclic variation in immune responsiveness observed in female rats.

The gonadal hormone effects in the morphology of the thymus are also well known (24,35,36,48) and are sup- ported hy the present study. After castration there is an increase in the mass of peripheral lymph nodes, spleen, ::nd thymas !see 36 and Fig. 5). Along with the increase iii the thymic mass induced by castration, there are structural alterations in the thymus (11‘ castrated ani mals. The size of the cortex is increased, and cellular density is greater. Since castration leads to an increase in the number of glucocorticoid-sensitive (cortical) cells, while the number of cortisone-resistant (medullary) cells remains unchanged, it has been suggest,ed that cas- tration may lead to a greater production of thymic lym- phocytes originating from precursor cells (see 36).

This seems of particular interest, in view of the selec- tive localization of the thymic p-adrenergic receptor in the medulla suggesting that the marked loss of receptors in the medullary region is indeed a specific effect of go-

nadal hormone withdrawal. On the other hand, estro- gen-treated rats have gross alterations of thymic tissue architecture. Our study is in agreement with other inves- tigations showing atrophy of the lobules, increased fat content of the gland, and destruction of the thymic lym- phocytes (36). The specific effect of estradiol in the med- ullar B-adrenergic receptor population is again observed, since treatment with pharmacological doses of the ste- roid has a potent stimulatory effect on receptor density which appears morphologically distributed throughout, the organ due to the atrophy of the cortical thymic com- partment (see Fig. 3C).

That steroid hormones can modulate tissue sensitivity in a variety of systems and alter the adrenergic response, possibly through changes in the number of adrenergic receptors, is a well-known phenomenon (see 48). Such regulatory effects of sex steroids include modulation at the level of the brain (49), uterus (46,50,51), ovary (25, 52,53), prostate (54), and mammary gland (26,44), and a growing body of experimental evidence supports a physiological role played by the direct catecholaminergic innervation of these organs in the physiological regula- tion of the function of various endocrine glands (see 25, 26,53-56).

Steroid hormones are heterologous regulators of p-ad- renergic receptors; they can both upregulate the receptor and increase its coupling to the stimulatory guanyl nu- cleotide binding protein (see 46). Such steroid-induced upregulation is, however, organ- and cell-specific (see 46); further studies are required to ascertain whether the effect of estradiol at the thymus level is affecting the transcription of the genes for the adrenergic receptor, thus influencing the steady-state level of the receptor in cells and/or modulating the coupling of the receptor with other components of the adenylate cyclase system (see 46, 57).

In summary, we have shown profound modifications in both organization and density of the oz-adrenergic re- ceptor population of the rat thymus gland following different hormonal manipulations. This suggests that a complex and potentially import,ant local regulatory net- work involving catecholamines, sex steroids, and possi- bly thymic hormones and neuropeptides participates in I hr dynamic* cant rol of the d-adrenergic receptor, thus providing a morphological substrate for direct interac- tion of catecholamines with other factors in the control of thymus gland development, differentiation, and func- tion.

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